CN110108874A - Quickly colloid gold immune analysis test paper item, preparation and its application of detection cyclopiazonic acid - Google Patents
Quickly colloid gold immune analysis test paper item, preparation and its application of detection cyclopiazonic acid Download PDFInfo
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Abstract
The invention discloses colloid gold immune analysis paper slip, preparation and its applications of a kind of quickly detection cyclopiazonic acid.It includes bottom plate, the one side of bottom plate successively pastes water absorption pad, detecting pad, gold-labelled pad and sample pad from top to bottom, adjacent each pad is in the overlapping connection in junction, the detecting pad is using nitrocellulose filter as base wad, lateral nature controlling line and detection line are set from top to bottom on nitrocellulose filter, the detection line is coated with cyclopiazonic acid-ovalbumin conjugate, and nature controlling line is coated with rabbit-anti mouse polyclonal antibody；The gold-labelled pad is laterally coated with the anti-cyclopiazonic acid monoclonal antibody of nano gold mark, and the anti-cyclopiazonic acid monoclonal antibody is generated by the hybridoma cell strain YTT-2 that deposit number is CCTCC NO:C201871.The test strips have the characteristics that detect quick, easy to operate, high sensitivity for detecting cyclopiazonic acid.
Description
Technical field
The invention belongs to field of biological detection, and in particular to the quickly colloid gold immune analysis test paper of detection cyclopiazonic acid
Item, preparation and its application.
Background technique
Cyclopiazonic acid is mainly the secondary metabolite generated by aspergillus flavus and aspergillus parasiticus secretion, is that a kind of can cause
The natural toxic compounds of the various damages of people and animals.Cyclopiazonic acid is widely present in the agricultural product such as rice, corn, peanut, feed
In the food such as cheese, after contaminated food products and feed, the health and lives of people and animals can be threatened to pacify directly or indirectly to food chain
Entirely.Therefore reinforce detection, particularly speed to cyclopiazonic acid in agricultural product and food to survey, to understand and grasp food in time
And the health information of feed.
Cyclopiazonic acid detection technique commonly used in the prior art mainly has thin layer chromatography (TLC), high performance liquid chromatography
Method (HPLC), immune analysis method.The sample pre-treatments step time and effort consuming of first two method, instrument price is expensive, and needs
The disadvantage of professional technician etc..The shortcomings that immune analysis method overcomes the above two has high specificity, high sensitivity, sample
The advantages that product pre-treatment is simple, at low cost, small to the contamination hazard of experimenter and environment.Most common immune analysis method has
Enzyme linked immunosorbent assay, nanogold immunochromatographic method etc..There is presently no come out for cyclopiazonic acid immuno-chromatographic test paper strip.
Therefore, research establish it is a kind of for cyclopiazonic acid immuno-chromatographic test paper strip for cyclopiazonic acid content detection have it is very heavy
The meaning and application value wanted.
Summary of the invention
The technical problem to be solved by the present invention is to provide quickly detection ring in view of the deficiency of the prior art
Colloid gold immune analysis test paper item, preparation and its application of cyclopiazonic acid.The test strips have inspection for detecting cyclopiazonic acid
The characteristics of survey is quickly, easy to operate, high sensitivity.
The present invention to solve above-mentioned technical problem used by technical solution are as follows:
The quickly colloid gold immune analysis test paper item of detection cyclopiazonic acid, including bottom plate, the one side of bottom plate is from top to bottom
Water absorption pad, detecting pad, gold-labelled pad and sample pad are successively pasted, adjacent each pad is in the overlapping connection in junction, and the detecting pad is with nitre
Acid cellulose film is base wad, and nature controlling line and detection line, the detection line coating is laterally arranged on nitrocellulose filter from top to bottom
There is cyclopiazonic acid-ovalbumin conjugate (CPA-OVA), nature controlling line is coated with rabbit-anti mouse polyclonal antibody；The gold-labelled pad
It is laterally coated with the anti-cyclopiazonic acid monoclonal antibody of nano gold mark, the anti-cyclopiazonic acid monoclonal antibody is by preservation
The hybridoma cell strain YTT-2 that number is CCTCC NO:C201871 is generated.
According to the above scheme, the water absorption pad long 16~18mm, wide 2~4mm；Detecting pad grow 25~30mm, it is wide by 2~
4mm；Gold-labelled pad grows 6~9mm, wide 2~4mm；Sample pad grow 12~18mm, wide 2~4mm, the adjacent overlapping length respectively padded be 1~
3mm。
According to the above scheme, the water absorption pad is blotting paper；The bottom plate is cardboard.
According to the above scheme, the spacing on edge is 15~20mm, matter on the detection line on the detecting pad and nitrocellulose filter
The spacing for controlling line and detection line is 5~10mm.
According to the above scheme, cyclopiazonic acid-ovalbumin conjugate needed for detection line per cm on the detecting pad
(CPA-OVA) package amount is 80~400ng；The package amount of rabbit-anti mouse polyclonal antibody needed for nature controlling line per cm is 100
~300ng.
According to the above scheme, the partial size of nanogold used in the gold-labelled pad is 15~20nm；Every li in the gold-labelled pad
The dosage of the anti-cyclopiazonic acid monoclonal antibody of nano gold mark needed for rice spraying length is 100~200ng.
The preparation method of the colloid gold immune analysis paper slip of quickly detection cyclopiazonic acid as described above, including following step
It is rapid:
(1) preparation of water absorption pad
Blotting paper is cut out into obtain water absorption pad；
(2) preparation of detecting pad
The coating of detection line:
By cyclopiazonic acid-ovalbumin conjugate with coating buffer at the coating buffer of 0.1~0.5mg/mL；In
Along the position for being 15~20mm on nitrocellulose filter, coating buffer is laterally coated in nitrocellulose filter with line spray mode
On obtain detection line, in detection line per cm required cyclopiazonic acid-ovalbumin conjugate package amount be 80~400ng,
Then 60~120 minutes dry under the conditions of 37~40 DEG C；
The coating of nature controlling line:
By rabbit-anti mouse polyclonal antibody with coating buffer at the coating buffer of 0.5mg/mL；In away from detection line 5~
Coating buffer is laterally coated on nitrocellulose filter with line spray mode, obtains nature controlling line, nature controlling line per cm by the position of 10mm
The package amount of rabbit-anti mouse polyclonal antibody needed for upper is 100~300ng, then 60~120 points dry under the conditions of 37~40 DEG C
Clock；
(3) preparation of sample pad
Glass fibre membrane is put into confining liquid and is soaked, is taken out, it is 10~16 hours dry under the conditions of 37~40 DEG C, obtain sample
Then product pad sets room temperature preservation in drier；
(4) preparation of gold-labelled pad
Glass fibre membrane is put into confining liquid and is soaked, is taken out, it is 10~16 hours dry under the conditions of 37~40 DEG C, in
On dry glass fibre membrane, the anti-cyclopiazonic acid monoclonal antibody solution of nano gold mark is laterally carried out with spray mode
It sprays, the anti-cyclopiazonic acid monoclonal antibody of nano gold mark needed for spraying length per cm is 100~200ng, then very
2~6h of vacuum freecing-dry sets room temperature preservation in drier；The anti-cyclopiazonic acid monoclonal antibody is by deposit number
The hybridoma cell strain YTT-2 of CCTCC NO:C201871 is generated；
(5) assembling of test strips
Water absorption pad, detecting pad, gold-labelled pad and sample pad are successively pasted from top to bottom in the one side of bottom plate, and adjacent each pad is even
The colloid gold immune that the overlapping connection in place is connect to get quick detection cyclopiazonic acid analyzes paper slip.
According to the above scheme, coating buffer used in the detection line coating is to prepare to obtain by the following method
: 1g bovine serum albumin(BSA), 0.02g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g chlorination
Potassium, 0.02g potassium dihydrogen phosphate add water to be settled to obtained by 100mL；
Coating buffer used in the coating of the nature controlling line are as follows: 1g ovalbumin, 0.02g sodium azide,
0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate add water to be settled to 100mL
Gained；
According to the above scheme, the confining liquid configures obtain by the following method: by 1~2g ovalbumin, 2~5g
Sucrose, 0.02~0.05g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, 0.02g
Potassium dihydrogen phosphate adds water to be settled to obtained by 100mL；
According to the above scheme, the anti-cyclopiazonic acid monoclonal antibody solution of the nano gold mark is using unsaturated label
Method preparation, method particularly includes: taking 50.0mL mass concentration is 0.01% nano-Au solution, with 0.1mL0.1mol/L carbon
Sour aqueous solutions of potassium adjusts pH；It is slowly added to the anti-cyclopiazonic acid monoclonal antibody water of 2mL 0.1mg/mL while stirring
Solution continues to stir 30min；The whole mass concentration that mass concentration is 10% ovalbumin aqueous solution to ovalbumin, which is added, is
1%, continue to stir 30min；After 4 DEG C of placement 2h, 3000r/min is centrifuged 15min, takes supernatant, abandons precipitating；By supernatant
12000r/min is centrifuged 30min, discards supernatant liquid, and 50.0mL label washing is added and saves liquid；It is centrifuged again with 12000r/min
30min discards supernatant liquid, and precipitating is saved liquid with label washing and is resuspended, 5.0mL concentrate is obtained, it is spare to set 4 DEG C of refrigerators；
The label washing preservation liquid is prepared by the following method to be obtained: 2.0g polyethylene glycol-20000,0.2g
Sodium azide, 0.1235g boric acid, pure water are settled to 1000mL, 0.22 μm of membrane filtration gained；
The application of the colloid gold immune analysis paper slip of quickly detection cyclopiazonic acid as described above, the method is as follows: by sample
Product are extracted through methanol, obtain methanol extract liquid, methanol extract liquid is diluted with water, make methanol in dilution final concentration of 20~
30%, sample solution is obtained, takes the sample solution diluted to be added dropwise one as detection liquid and quickly detects cyclopiazonic acid
Colloid gold immune analyzes the sample pad of paper slip, as test strip, while taking isometric methanol-water as negative right
According to liquid, the sample pad of the colloid gold immune analysis paper slip of another quick detection cyclopiazonic acid is added dropwise, as control
Test strips read result after a period of time；
Testing result:
It is positive: when the nature controlling line of test strip shows red lines, and detection line does not develop the color, to show sample to be tested
In cyclopiazonic acid total content be greater than or equal to 5ng/mL；
When nature controlling line shows red lines, and detect line color it is more of light color than control test strips detection line when, show to
Cyclopiazonic acid content is equal to or higher than 1ng/mL and is lower than 5ng/mL in sample solution；
It is negative: when the nature controlling line of test strip shows red lines, and to detect line color and control stripes item is examined
When survey line color is close, negative findings are judged to, show the total content of the cyclopiazonic acid in sample to be tested lower than 1ng/mL；
Invalid: when nature controlling line does not develop the color, no matter the detection line of test strip shows or is not displayed in red lines, the examination
Paper slip is judged in vain；
Most afterwards through converting up to the content of cyclopiazonic acid in sample to be tested.
According to the above scheme, the methanol extracts are as follows: and sample to be tested is levigate, the methanol that addition volumetric concentration is 70%/
2% NaHCO310% chlorination is added with lower layer's water phase is collected after n-hexane extraction in aqueous solution, mechanical shaking extraction, centrifuging and taking supernatant
Potassium solution, and it is adjusted to pH2-3 with HCl, with chloroform mechanical shaking extraction, lower layer's chloroform layer is collected, rotary evaporation concentration removes chloroform,
Precipitating is dissolved with methanol, obtains methanol extract liquid.
The working principle of the colloid gold immune analysis paper slip of the quick detection cyclopiazonic acid: when testing sample solution is added
When on to the sample pad of test strips lower end, testing sample solution is moved along test strips to water absorption pad direction through capillary action, when
When it is moved to gold-labelled pad, the anti-cyclopiazonic acid monoclonal antibody of nano gold mark is dissolved.When in sample contain ring Ah
When Buddhist nun's acid, cyclopiazonic acid will be combined with the anti-cyclopiazonic acid monoclonal antibody of the nano gold mark in gold-labelled pad and one is in the same direction
Upper swimming, when reaching the detection line of fixed antigen, antigen is by the anti-ring with cyclopiazonic acid competitive binding nano gold mark
Limited antigen binding site in cyclopiazonic acid monoclonal antibody, cyclopiazonic acid content is higher in sample, anti-in detection line
The anti-cyclopiazonic acid monoclonal antibody for the nano gold mark that proper energy enough combines will be fewer, and the developed band color of formation is more shallow.When
When being less than certain quantity of the nano gold mark that antigen is combined, will not have red lines to occur at detection line.No matter sample
Whether cyclopiazonic acid is contained in product, the nano gold mark that the antigen on not tested survey line is intercepted and captured or nano gold mark anti-ring
The conjugate of cyclopiazonic acid monoclonal antibody and cyclopiazonic acid will move on nature controlling line and anti-with second on nature controlling line
Body rabbit-anti mouse polyclonal antibody rabbit-anti mouse polyclonal antibody is combined and is developed the color by enrichment, so cyclopiazonic acid is free of in sample,
It is two red stripes when as negative, i.e. nature controlling line and detection line is red；It is as positive containing a certain amount of cyclopiazonic acid
Property when there are two types of situation: 1, only there is a red nature controlling line, detection line does not develop the color；2, a red nature controlling line and one it is pale red
Color detection line；And nature controlling line does not have colour band appearance then to show that test strips fail.
The beneficial effects of the present invention are:
(1) cyclopiazonic acid content is detected.The colloid gold immune of quick detection cyclopiazonic acid provided by the invention is analyzed
The antibody that paper slip uses is that anti-cyclopiazonic acid monoclonal antibody is actually answered for detecting cyclopiazonic acid content high sensitivity
It is big with value.The lowest detection of the colloid gold immune analysis test paper item of quick detection cyclopiazonic acid provided by the invention is limited to
1ng/mL。
(2) easy to operate.It is only needed when being detected with the immuno-chromatographic test paper strip of the quick detection cyclopiazonic acid content
Sample extracting solution is added dropwise in the sample pad of test strips, is operated for single step, does not need professional, it is easy to operate
It is convenient.
(3) detection process does not need cyclopiazonic acid standard solution as positive control.Quick detection provided by the invention
When the immuno-chromatographic test paper strip test sample of cyclopiazonic acid content, do not need to use cyclopiazonic acid standard solution as the positive
Control, and only need to be with water as negative control, so as to avoid the secondary pollution of cyclopiazonic acid.
Detailed description of the invention
Fig. 1 and Fig. 2 is the structural representation of the colloid gold immune analysis test paper item of quick detection cyclopiazonic acid of the invention
Figure.In figure: in figure: 1 bottom plate, 2 water absorption pads；3 detecting pads；4 gold-labelled pads；5 sample pads；6 nature controlling lines；7 detection lines.
Fig. 3 is the result judgement figure of 1# corn powder sample in embodiment 2.In figure: a control stripes item；B Test paper
Item.
The result judgement figure of 2# corn powder sample in Fig. 4 embodiment 2.In figure: a control stripes item；B test strip.
Specific embodiment
Embodiment 1: the preparation of anti-cyclopiazonic acid monoclonal antibody
The hybridoma cell strain YTT-2 that anti-cyclopiazonic acid monoclonal antibody is CCTCC NO.C C201871 by deposit number
It generates.It is specific as follows:
Cyclopiazonic acid monoclonal antibody hybridoma cell strain YTT-2 is injected intraperitoneally in advance at incomplete Freund's adjuvant
In the BALB/c mouse body managed, the ascites of mouse is collected, using caprylic acid-ammonium antibody purification, concrete operations are as follows: use double
Layer filter paper filters mouse ascites, and for filtered ascites in 4 DEG C, 12000r/min is centrifuged 15min or more, supernatant is drawn, by supernatant
It is mixed with the acetate buffer of 4 times of volumes, is slowly added to caprylic acid while stirring, caprylic acid volume needed for every milliliter of ascites
For 30~35 μ L, 30~60min of mixed at room temperature, 4 DEG C of standing 2h or more, then 4 DEG C, 12000r/min is centrifuged 30min or more,
Abandon precipitating, after obtained supernatant is filtered with double-layer filter paper, be added 1/10 filtrate volume molar concentration be 0.1mol/L and
The phosphate buffer of pH7.4 adjusts the pH to 7.4 of the mixed liquor with the sodium hydroxide solution of 2mol/L, and 4 DEG C are pre-chilled, slowly
Ammonium sulfate is added to the final concentration of 0.277g/mL of ammonium sulfate, 4 DEG C of standing 2h or more, then 4 DEG C, 12000r/min is centrifuged
30min or more abandons supernatant, and gained is precipitated and is resuspended with the 0.01mol/L phosphate buffer of former ascites volume 1/10, is packed into saturating
Bag is analysed, is dialysed with pure water, the protein solution sufficiently dialysed is set into -70 DEG C of refrigerator freezings, is then frozen with frozen vacuum dryer
It is dry, freeze-dried powder is collected to get purified anti-cyclopiazonic acid monoclonal antibody, antibody is set spare in -20 DEG C of refrigerators；
The acetate buffer is 0.29g sodium acetate, and 0.141mL acetic acid adds water to be settled to obtained by 100mL；Described
The phosphate buffer of 0.01mol/L is 0.9g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, di(2-ethylhexyl)phosphate
Hydrogen potassium 0.02g adds water to be settled to obtained by 100mL.
With the anti-cyclopiazonic acid monoclonal antibody of commercially available subtype identification kit identification hybridoma cell strain YTT-2 secretion
Hypotype be IgG2a type.
With the conventional non-effect for connecing competitive enzyme-linked immune adsorption analysis (ELISA) method and measuring the mouse hydroperitoneum antibody of RGDV of YTT-2 indirectly
Valence is up to 1.2 × 105, i.e. mouse hydroperitoneum antibody of RGDV dilution 1.2 × 105Times when solution measurement result be the positive.Conventional indirect competition
ELISA method identifies its sensitivity (IC to cyclopiazonic acid50) it is 0.84ng/mL, to aflatoxin B1, B2, G1, G2, M1
0.1% is respectively less than with the cross reacting rate of sterigmatocystin.
The screening of hybridoma cell strain YTT-2:
1. antigen synthesis and animal immune
It buys commercially available cyclopiazonic acid standard items and carries out comlete antigen synthesis, specific synthesis step is as follows: by 1mg CPA
It is dissolved in 1mL0.05M NaHCO350% methanol aqueous solution in；Take 2mg hemocyanin (KLH) that 0.4ml3M sodium acetate is added,
0.2mL formaldehyde is added dropwise under the conditions of being stirred at room temperature, in 1min, persistently stirs 10min；CPA is slowly added into KLH dropwise
In, 16h or more is persistently stirred under room temperature.Final reacting product CPA-KLH is placed in suitable size bag filter, in PBS
In 4 DEG C whisk dialysis three days.Same method synthesis detection original CPA-OVA.
6 week old female BaLb/c mouse 6 is bought, the immune cyclopiazonic acid comlete antigen CPA-KLH voluntarily synthesized exempts from
Epidemic disease dosage is 100 μ g/.It is immunized for the first time by comlete antigen CPA-KLH and Freund's complete adjuvant mixing and emulsifying, carries out back skin
Lower multi-point injection is immune.Just exempt from interval 3 weeks, every minor tick is immunized for 2 weeks later, is emulsified and is carried out using incomplete Freund's adjuvant
It is immune.It is immune after a week from third time, tail vein blood is carried out, serum is separated, it is anti-using indirect elisa method monitoring mice serum
Body potency measures mice serum sensitivity with indirect competitive ELISA method, selects potency, the relatively higher serum pair of sensitivity
The mouse answered carries out last time spurt and is immunized, and fusion takes 100 μ g immunogenes to be dissolved in 200 μ L PBS direct injection abdominal cavities in first 3 days.
Freund's adjuvant is purchased from Sigma-Aldrich company.
2. cell fusion
After last time is made a spurt immune 3 days, use 50% (weight percent) polyethylene glycol i.e. PEG (molecular weight for
1450) make fusion agent, carry out cell fusion according to a conventional method, the specific steps are as follows:
Cervical dislocation puts to death immune mouse, aseptically wins spleen, separating Morr. cell is ground, with source of mouse marrow
Oncocyte SP2/0, than mixing, is washed cell mixing with RPMI-1640 basic culture solution, is merged with 50%PEG by 5:1 number, is merged
1 minute, RPMI-1640 basic culture solution is then filled it up with, is centrifuged, removes supernatant, mouse boosting cell and source of mouse myeloma cell
The fused cell that SP2/0 is formed is resuspended with 72mLRPMI-1640 basic culture solution, and the cell that gets up will be resuspended, and to be added drop-wise to 96 holes thin
In born of the same parents' culture plate, 2 drops/hole are set 37 DEG C of carbon dioxide incubators and are supported, and the RPMI-1640 basic culture solution is to contain 20%
(percentage by volume) fetal calf serum, 2% (weight percent) growth factor and 1% (weight percent) hypoxanthine-amino butterfly
Purine-thymidine, that is, HAT.Above-mentioned SP2/0 is purchased from ingression Ke Biotechnology Co., Ltd；The culture of the basis RPMI-1640
Liquid is purchased from Hyclone company；It is public that 1% hypoxanthine-aminopterin-thymidine, that is, HAT is purchased from Sigma- Aldrich
Department.
3. the screening of cell strain and clone are the 12nd day or so after cell fusion, cell colony, which is grown to, accounts for 1/2 area of bottom hole
Size, culture solution turn yellow, and can carry out antibody test.The culture hole for having Growth of Hybridoma Cell is carried out using ELISA method
Screening, screening are carried out in two steps, and the first step filters out anti-cyclopiazonic acid without anti-carrier egg using indirect non-competing ELISA method
The positive hole of white KLH；Second step detects the positive hole that the first step filters out using indirect competitive ELISA method, with ring
Cyclopiazonic acid is former as competition, and selecting the higher hole of light absorption value and sensitivity, (light absorption value is higher to refer to that competition was originally i.e. positive for 0 hole
The final tested volume of control wells is higher, the higher competition original content that is, IC referred to when inhibiting rate is 50% of sensitivity50It is worth smaller),
It is cloned, is detected using same two-step method within 10 days or so after clone, such repeated cloning 2-3 using limiting dilution assay
After secondary, hybridoma cell strain YTT-2 being obtained, China typical culture collection center (CCTCC) is preserved in, preservation address is, in
State, Wuhan, Wuhan University, deposit number are CCTCC NO.C201871, and the deposit date is on March 23rd, 2018.
The anti-cyclopiazonic acid monoclonal antibody hybridoma cell antibody variable sequences strain YTT-2 measurement
(1) extract total serum IgE: using Tiangeng company total RNA extraction reagent box and to specifications extraction can produce hybridization
The total serum IgE of tumor cell strain YTT-2.
(2) synthesize cDNA: the total serum IgE obtained using step 1 is template, oligo (dT)15For primer, according to
SuperScriptTM- 2II reverse transcriptase specification carries out reverse transcription, synthesizes the first chain of cDNA；Primer oligo (dT)15By
Invitrogen is bought；
(3) PCR method clones variable region gene: being drawn according to the conserved positions design of mouse antibody gene sequence in GENBANK
Object expands heavy chain of antibody, light-chain variable region gene by template of CDNA.PCR program are as follows: 94 DEG C of 30s, 55 DEG C of 50s, 72 DEG C
1min expands 30 circulations, last 72 DEG C of extensions 10min.Ago-Gel electricity of the PCR product Jing Guo 1% (weight percent)
After swimming separation, DNA fragmentation is recycled with kits, is connected in carrier pMD18-T, bacillus coli DH 5 alpha competence is converted
Cell, picking positive colony send to Suzhou Hong Xun Biotechnology Co., Ltd and are sequenced.Wherein the sequence of primer is respectively as follows:
Heavy chain variable region primer is 5 '-AGG TSM ARC TGC AGS AGT CWG G-3 ' (22mer) and 5 '-TGA GGA
Wherein S, M, R and W are to annex base, M=A/C, R=to GACGGT GAC CGT GGT CCC TTG GCC CC-3 ' (32mer)
A/G, S=C/G, W=A/T, light chain variable region primer are 5 '-GAC ATT GAG CTC ACC CAG CTT GGT GCC-3 '
(24mer) and 5 '-CCG TTT CAG CTC CAG CTT GGT CCC-3 ' (24mer).
Obtained gene order result: the long 360bp of heavy chain variable region coding gene sequence, sequence such as SEQ ID NO:1 institute
Show, derives that the encoded heavy chain variable region of the gene order is made of 120 amino acid according to gene order obtained, sequence
Column are as shown in SEQ ID NO:3.The long 322bp of light chain variable region coding gene sequence, sequence as shown in SEQ ID NO:2, according to
Gene order obtained derives that the encoded light chain variable region of the gene order is made of 107 amino acid, sequence such as SEQ
Shown in ID NO:4.
Embodiment 2:
The quickly colloid gold immune analysis test paper item of detection cyclopiazonic acid, including bottom plate 1, the one side of bottom plate is from top to bottom
Water absorption pad 2, detecting pad 3, gold-labelled pad 4 and sample pad 5 are successively pasted, adjacent each pad is in the overlapping connection in junction, the detecting pad
Using nitrocellulose filter as base wad, nature controlling line 6 and detection line 7, the detection are laterally set from top to bottom on nitrocellulose filter
Line is coated with cyclopiazonic acid-ovalbumin conjugate (CPA-OVA), and nature controlling line is coated with rabbit-anti mouse polyclonal antibody；It is described
Gold-labelled pad is laterally coated with the anti-cyclopiazonic acid monoclonal antibody of nano gold mark, the anti-cyclopiazonic acid monoclonal antibody
It is generated by the hybridoma cell strain YTT-2 that deposit number is CCTCC NO:C201871.
The quickly preparation method of the colloid gold immune analysis test paper item of detection cyclopiazonic acid, comprising the following steps:
(1) preparation of water absorption pad
Blotting paper is cut into 16mm up to water absorption pad；
(2) preparation of detecting pad
The coating of detection line:
Cyclopiazonic acid-ovalbumin conjugate is configured to the coating buffer of 0.5mg/mL, in apart from nitrocellulose filter
Upper edge is the position of 15mm, is laterally coated on nitrocellulose filter coating buffer with line spray mode and obtains detection line, per cm
Required cyclopiazonic acid-ovalbumin conjugate package amount is 350ng in detection line, then dry 120 under the conditions of 37 DEG C
Minute；
The coating of nature controlling line:
Rabbit-anti mouse polyclonal antibody is configured to the coating buffer of 0.5mg/mL；In the position away from detection line 5mm, with line spray side
Coating buffer is laterally coated on nitrocellulose filter by formula, obtains nature controlling line, and required rabbit-anti mouse is polyclonal on nature controlling line per cm
The package amount of antibody is 200ng, then 120 minutes dry under the conditions of 37 DEG C；
(3) preparation of sample pad:
Glass fibre membrane is put into confining liquid and is soaked, is taken out, it is 16 hours dry under the conditions of 37 DEG C, sample pad is obtained, so
Room temperature preservation in postposition drier；
(4) preparation of gold-labelled pad:
Glass fibre membrane is put into confining liquid and is soaked, is taken out, it is 16 hours dry under the conditions of 37 DEG C, in the glass dried
On glass tunica fibrosa, the anti-cyclopiazonic acid monoclonal antibody solution of nano gold mark is laterally sprayed with spray mode, often
The anti-cyclopiazonic acid monoclonal antibody of nano gold mark needed for centimetre spraying length is 150ng, then vacuum freeze drying 6h,
Set room temperature preservation in drier；The anti-cyclopiazonic acid monoclonal antibody is the miscellaneous of CCTCC NO:C201871 by deposit number
Tumor cell strain YTT-2 is handed over to generate；
(5) assembling of test strips
Water absorption pad, detecting pad, gold-labelled pad and sample pad are successively pasted from top to bottom in the one side of cardboard, and adjacent each pad is even
The overlapping connection in place is connect, overlapping length is high-sensitivity immunochromatographitest test strip of the 1mm to get quick detection cyclopiazonic acid；
Coating buffer used in the coating buffer of the envelope antigen are as follows: 1g bovine serum albumin(BSA), 0.02g Azide
Sodium, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate add water to be settled to
Obtained by 100mL；
Coating buffer used in the coating buffer of the coating rabbit-anti mouse polyclonal antibody are as follows: 1g ovalbumin,
0.02g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate,
Water is added to be settled to obtained by 100mL；
The confining liquid configures obtain by the following method: by 1g ovalbumin, 2g sucrose, 0.02g Azide
Sodium, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate add water to be settled to
Obtained by 100mL；
The anti-cyclopiazonic acid monoclonal antibody solution of the nano gold mark is prepared using unsaturated labelling method,
Method particularly includes: taking 50.0mL commercial available quality concentration is 0.01% nano-Au solution, water-soluble with 0.1mL0.1mol/L potassium carbonate
Liquid adjusts pH；；It is slowly added to the anti-cyclopiazonic acid monoclonal antibody aqueous solution of 2mL0.1mg/mL while stirring, after
Continuous stirring 30min；It is 1% that the whole mass concentration that mass concentration is 10% ovalbumin aqueous solution to ovalbumin, which is added, is continued
Stir 30min；After 4 DEG C of placement 2h, 3000r/min is centrifuged 15min, takes supernatant, abandons precipitating；By supernatant 12000r/
Min is centrifuged 30min, discards supernatant liquid, and 50.0mL label washing is added and saves liquid；30min is centrifuged with 12000r/min again, is abandoned
Supernatant is removed, precipitating is saved into liquid with label washing and is resuspended, 5.0mL concentrate is obtained, it is spare to set 4 DEG C of refrigerators.
The 0.1mol/L wet chemical are as follows: 13.8g potassium carbonate is dissolved in pure water and is settled to 1000mL, 0.22 μm of filter
Film filtering gained；The label washing preservation liquid is prepared by the following method to be obtained: 2.0g polyethylene glycol-20000,
0.2g Sodium azide, 0.1235g boric acid, pure water are settled to 1000mL, 0.22 μm of membrane filtration gained.
The application of the colloid gold immune analysis paper slip of above-mentioned quick detection cyclopiazonic acid:
Each 5g of 1# and 2# corn powder sample is weighed respectively in 50mL centrifuge tube, and 20ml methanol/2%NaHCO is added3Water
Solution, mechanical shaking extraction 1h, centrifuging and taking supernatant.50mL n-hexane is added by several times, vibrates hybrid extraction, collects lower water after layering
Phase.5ml10% Klorvess Liquid is added, and is adjusted to pH2-3 with HCl.The oscillation mixing of 30mL chloroform is added by several times repeatedly to extract,
Lower layer's chloroform layer is collected, rotary evaporation is concentrated and 1mL methanol is used to dissolve, and 9mL sample diluting liquid is added and dilutes filtrate, mixes.It takes
The high sensitivity that the sample solution that 150 μ L have diluted is added dropwise to quickly detection cyclopiazonic acid respectively as detection liquid is immune
The sample pad of chromatograph test strip, as test strip；Result is read after 15 minutes；
Testing result: the nature controlling line of 1# corn powder sample detection test strips shows red lines, and detection line is not shown
Color is then judged to positive findings, shows that the content of the cyclopiazonic acid in sample to be tested is higher than 5ng/mL, sees Fig. 3；2# corn powder
The nature controlling line of sample detection test strips shows red lines, and detect line color than control test strips detection line it is of light color, then
Positive findings are judged to, shows that the content of the cyclopiazonic acid in sample to be tested is equal to or higher than 1ng/mL and is lower than 5ng/mL, sees
Fig. 4.
<110>Inst. of Oil Crops, Chinese Academy of Agriculture
<120>quickly colloid gold immune analysis test paper item, preparation and its application of detection cyclopiazonic acid
<160> 4
<210> 1
<211> 360bp
<212> DNA
<213>mouse
<400> 1
gagatccagc tgcagcagtc tggacctgac ctgatgaagc ctggggcttc 50
agtgaagata tcctgcaagg cttctggtta ctcattcact acctactaca 100
tgcactgggt gaagcagagc catggaaaga gccttgagtg gattggatat 150
attgatcctt tcaatggtga tactaggtac aacccgaaat tcaaggccaa 200
ggccacattg actgtagaca aatcttccag cacagcctac atgcagctca 250
gcagcctgac atctgaggac tctgcagtct attactgtgc aagagtttat 300
tactacggta gtagctggtt tgcttactgg ggccaaggga ctctggtcac 350
tgtctctgca 360
<210> 2
<211> 322bp
<212> DNA
<213>mouse
<400> 2
gacatcctga tgacccaatc tccatcctcc atgtctgtat ctctgggaga 50
cacagtcacc atcacttgcc atgcaagtca gggcattagc agtaatatag 100
ggtggttgca gcagaaacca gggaaatcat ttaagggcct gatctatcaa 150
ggaagcaact tggaagatgg agttccatca aggttcagtg gcagtggatc 200
tggagcagat tattctctca ccatcagcag cctggaatat gaagattttg 250
cagactatta ctgtgtacag tttgctcagt ttcctcccac gttcggtgct 300
gggaccaagc tggagctgaa ac 322
<210> 3
<211> 120
<212> PRT
<213>mouse
<400> 3
Glu Ile Gln Leu Gln Gln Ser Gly Pro Asp Leu Met Lys Pro Gly
1 5 10 15
Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr
20 25 30
Thr Tyr Tyr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu
35 40 45
Glu Trp Ile Gly Tyr Ile Asp Pro Phe Asn Gly Asp Thr Arg Tyr
50 55 60
Asn Pro Lys Phe Lys Ala Lys Ala Thr Leu Thr Val Asp Lys Ser
65 70 75
Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
80 85 90
Ser Ala Val Tyr Tyr Cys Ala Arg Val Tyr Tyr Tyr Gly Ser Ser
95 100 105
Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
110 115 120
<210> 4
<211> 107
<212> PRT
<213>mouse
<400> 4
Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Met Ser Val Ser Leu
1 5 10 15
Gly Asp Thr Val Thr Ile Thr Cys His Ala Ser Gln Gly Ile Ser
20 25 30
Ser Asn Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ser Phe Lys
35 40 45
Gly Leu Ile Tyr Gln Gly Ser Asn Leu Glu Asp Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile
65 70 75
Ser Ser Leu Glu Tyr Glu Asp Phe Ala Asp Tyr Tyr Cys Val Gln
80 85 90
Phe Ala Gln Phe Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu
95 100 105
Leu Lys
107
Claims (10)
1. the quickly colloid gold immune analysis test paper item of detection cyclopiazonic acid, it is characterised in that: including bottom plate, the one side of bottom plate
Water absorption pad, detecting pad, gold-labelled pad and sample pad are successively pasted from top to bottom, and adjacent each pad is in the overlapping connection in junction, the inspection
Pad is surveyed using nitrocellulose filter as base wad, lateral nature controlling line and detection line, the inspection are set from top to bottom on nitrocellulose filter
Survey line is coated with cyclopiazonic acid-ovalbumin conjugate, and nature controlling line is coated with rabbit-anti mouse polyclonal antibody；The gold-labelled pad is horizontal
To the anti-cyclopiazonic acid monoclonal antibody for being coated with nano gold mark, the anti-cyclopiazonic acid monoclonal antibody is compiled by preservation
Number for CCTCC NO:C201871 hybridoma cell strain YTT-2 generate.
2. the colloid gold immune analysis test paper item of quick detection cyclopiazonic acid according to claim 1, it is characterised in that:
The water absorption pad long 16~18mm, wide 2~4mm；Detecting pad grows 25~30mm, wide 2~4mm；Gold-labelled pad grows 6~9mm, wide by 2
~4mm；Sample pad grows 12~18mm, wide 2~4mm, and the adjacent overlapping length respectively padded is 1~3mm；The water absorption pad is water suction
Paper；The bottom plate is cardboard.
3. the colloid gold immune analysis test paper item of quick detection cyclopiazonic acid according to claim 1, it is characterised in that:
The spacing on edge is 15~20mm in detection line and nitrocellulose filter on the detecting pad, and the spacing of nature controlling line and detection line is
5~10mm.
4. the colloid gold immune analysis test paper item of quick detection cyclopiazonic acid according to claim 1, it is characterised in that:
Cyclopiazonic acid-ovalbumin conjugate package amount needed for detection line per cm is 80~400ng on the detecting pad；Often
The package amount of rabbit-anti mouse polyclonal antibody needed for centimetre nature controlling line is 100~300ng.
5. the colloid gold immune analysis test paper item of quick detection cyclopiazonic acid according to claim 1, it is characterised in that:
The partial size of nanogold used in the gold-labelled pad is 15~20nm；Nanometer needed for spraying length per cm in the gold-labelled pad
The dosage of the anti-cyclopiazonic acid monoclonal antibody of gold label is 100~200ng.
6. the preparation method of the colloid gold immune analysis paper slip of quick detection cyclopiazonic acid described in claim 1, feature
It is: the following steps are included:
(1) preparation of water absorption pad
Blotting paper is cut out into obtain water absorption pad；
(2) preparation of detecting pad
The coating of detection line:
By cyclopiazonic acid-ovalbumin conjugate with coating buffer at the coating buffer of 0.1~0.5mg/mL；In distance
Along the position for being 15~20mm on nitrocellulose filter, coating buffer is laterally coated on nitrocellulose filter with line spray mode and is obtained
To detection line, required cyclopiazonic acid-ovalbumin conjugate package amount is 80~400ng in detection line per cm, then
It is 60~120 minutes dry under the conditions of 37~40 DEG C；
The coating of nature controlling line:
By rabbit-anti mouse polyclonal antibody with coating buffer at the coating buffer of 0.5mg/mL；In away from 5~10mm's of detection line
Coating buffer is laterally coated on nitrocellulose filter by position with line spray mode, obtains nature controlling line, on nature controlling line per cm needed for
The package amount of rabbit-anti mouse polyclonal antibody is 100~300ng, then 60~120 minutes dry under the conditions of 37~40 DEG C；
(3) preparation of sample pad
Glass fibre membrane is put into confining liquid and is soaked, is taken out, it is 10~16 hours dry under the conditions of 37~40 DEG C, obtain sample
Pad, then sets room temperature preservation in drier；
(4) preparation of gold-labelled pad
Glass fibre membrane is put into confining liquid and is soaked, is taken out, dry 10~16 hours under the conditions of 37~40 DEG C, in having dried
Glass fibre membrane on, the anti-cyclopiazonic acid monoclonal antibody solution of nano gold mark is laterally sprayed with spray mode
It applies, the anti-cyclopiazonic acid monoclonal antibody of nano gold mark needed for spraying length per cm is 100~200ng, then vacuum
It is freeze-dried 2~6h, sets room temperature preservation in drier；The anti-cyclopiazonic acid monoclonal antibody is CCTCC by deposit number
The hybridoma cell strain YTT-2 of NO:C201871 is generated；
(5) assembling of test strips
Water absorption pad, detecting pad, gold-labelled pad and sample pad are successively pasted from top to bottom in the one side of bottom plate, and adjacent each pad is in junction
Overlapping connection analyzes paper slip to get the colloid gold immune of quick detection cyclopiazonic acid.
7. the preparation method of the colloid gold immune analysis paper slip of quick detection cyclopiazonic acid according to claim 6,
Be characterized in that: coating buffer used in the detection line coating is prepared by the following method to be obtained: 1g cow's serum
Albumin, 0.02g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, 0.02g di(2-ethylhexyl)phosphate
Hydrogen potassium adds water to be settled to obtained by 100mL；
Coating buffer used in the coating of the nature controlling line are as follows: 1g ovalbumin, 0.02g sodium azide, 0.8g chlorine
Change sodium, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate add water to be settled to obtained by 100mL；
The confining liquid configures obtain by the following method: by 1~2g ovalbumin, 2~5g sucrose, 0.02~
0.05g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate add
Water is settled to obtained by 100mL.
8. the preparation method of the colloid gold immune analysis paper slip of quick detection cyclopiazonic acid according to claim 6,
Be characterized in that: the anti-cyclopiazonic acid monoclonal antibody solution of the nano gold mark is prepared using unsaturated labelling method,
Its method particularly includes: taking 50.0mL mass concentration is 0.01% nano-Au solution, water-soluble with 0.1mL 0.1mol/L potassium carbonate
Liquid adjusts pH；It is slowly added to the anti-cyclopiazonic acid monoclonal antibody aqueous solution of 2mL 0.1mg/mL while stirring, after
Continuous stirring 30min；It is 1% that the whole mass concentration that mass concentration is 10% ovalbumin aqueous solution to ovalbumin, which is added, is continued
Stir 30min；After 4 DEG C of placement 2h, 3000r/min is centrifuged 15min, takes supernatant, abandons precipitating；By supernatant 12000r/min
It is centrifuged 30min, discards supernatant liquid, 50.0mL label washing is added and saves liquid；30min is centrifuged with 12000r/min again, is discarded
Precipitating is saved liquid with label washing and is resuspended, obtained 5.0mL concentrate, it is spare to set 4 DEG C of refrigerators by clear liquid；The label washing
Preservation liquid is prepared by the following method to be obtained: 2.0g polyethylene glycol-20000,0.2g Sodium azide, 0.1235g boric acid, pure water
It is settled to 1000mL, 0.22 μm of membrane filtration gained.
9. the application of the colloid gold immune analysis paper slip of quick detection cyclopiazonic acid described in claim 1, it is characterised in that:
Application method is as follows: sample being extracted through methanol, methanol extract liquid is obtained, methanol extract liquid is diluted with water, is made in dilution
Final concentration of the 20~30% of methanol, obtain sample solution, and the sample solution diluted is taken to be added dropwise one fastly as detection liquid
The sample pad of the colloid gold immune analysis paper slip of speed detection cyclopiazonic acid, as test strip, while taking isometric
Methanol-water as negative controls, the sample of the colloid gold immune analysis paper slip of another quick detection cyclopiazonic acid is added dropwise
Product pad reads result as control stripes item after a period of time；
Testing result:
It is positive: when the nature controlling line of test strip shows red lines, and detection line does not develop the color, to show in sample to be tested
The total content of cyclopiazonic acid is greater than or equal to 5ng/mL；
When nature controlling line shows red lines, and detect line color it is more of light color than control test strips detection line when, show to test sample
Cyclopiazonic acid content is equal to or higher than 1ng/mL and is lower than 5ng/mL in product solution；
It is negative: when the nature controlling line of test strip shows red lines, and to detect line color and control stripes detection line
When color is close, negative findings are judged to, show the total content of the cyclopiazonic acid in sample to be tested lower than 1ng/mL；
Invalid: when nature controlling line does not develop the color, no matter the detection line of test strip shows or is not displayed in red lines, the test strips
It is invalid to be judged to；
Most afterwards through converting up to the content of cyclopiazonic acid in sample to be tested.
10. the application of the colloid gold immune analysis paper slip of quick detection cyclopiazonic acid according to claim 9, feature
Be: the methanol extracts are as follows: and sample to be tested is levigate, methanol/2%NaHCO that volumetric concentration is 70% is added3It is water-soluble
With lower layer's water phase is collected after n-hexane extraction 10% Klorvess Liquid is added, and use HCl in liquid, mechanical shaking extraction, centrifuging and taking supernatant
It is adjusted to pH 2-3, with chloroform mechanical shaking extraction, collects lower layer's chloroform layer, rotary evaporation concentration removes chloroform, by precipitating methanol
Dissolution obtains methanol extract liquid.
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Citations (4)
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| CN1333837A (en) * | 1998-12-23 | 2002-01-30 | 诺维信公司 | Methods for producing polypeptides in aspergillus mutant cells |
| CN101930006A (en) * | 2010-08-05 | 2010-12-29 | 中国农业科学院油料作物研究所 | High-sensitivity immunochromatographic test strip for detecting total aflatoxin content quickly and preparation method thereof |
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| CN106932370A (en) * | 2017-03-07 | 2017-07-07 | 中国农业科学院油料作物研究所 | The immunochromatography time-resolved fluorescence kit of five kinds of mycotoxin composite pollutions such as synchronous detection AFB1 and application |
-
2019
- 2019-04-30 CN CN201910364421.5A patent/CN110108874B/en active Active
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|---|---|---|---|---|
| CN1333837A (en) * | 1998-12-23 | 2002-01-30 | 诺维信公司 | Methods for producing polypeptides in aspergillus mutant cells |
| CN101930006A (en) * | 2010-08-05 | 2010-12-29 | 中国农业科学院油料作物研究所 | High-sensitivity immunochromatographic test strip for detecting total aflatoxin content quickly and preparation method thereof |
| CN103217531A (en) * | 2013-04-03 | 2013-07-24 | 中国农业科学院油料作物研究所 | Immunochromatography test strip for synchronously detecting mixed pollution of aflatoxin, ochratoxin A and zearalenone, and preparation method |
| CN106932370A (en) * | 2017-03-07 | 2017-07-07 | 中国农业科学院油料作物研究所 | The immunochromatography time-resolved fluorescence kit of five kinds of mycotoxin composite pollutions such as synchronous detection AFB1 and application |
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| Title |
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| XUAN.HUANG AND FUN SUN.CHU: "Production and characterization of monoclonal and polyclonal antibodies against the mycotoxin cyclopiazonic acid", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 * |
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